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Black elderberry extract attenuates inflammation and metabolic
dysfunction indiet-induced obese mice
Nicholas J. Farrell, Gregory H. Norris, Julia Ryan, Caitlin M.
Porter, Christina Jiang andChristopher N. Blesso*Department of
Nutritional Sciences, University of Connecticut, Storrs, CT 06269,
USA
(Submitted 15 May 2015 – Final revision received 2 July 2015 –
Accepted 7 July 2015 – First published online 28 August 2015)
AbstractDietary anthocyanins have been shown to reduce
inflammation in animal models and may ameliorate obesity-related
complications. Blackelderberry is one of the richest sources of
anthocyanins. We investigated the metabolic effects of
anthocyanin-rich black elderberry extract(BEE) in a diet-induced
obese C57BL/6J mouse model. Mice were fed either a low-fat diet (n
8), high-fat lard-based diet (HFD; n 16),HFD+ 0·25 % (w/w) BEE
(0·25 %-BEE; n 16) or HFD+1·25 % BEE (1·25 %-BEE; n 16) for 16
weeks. The 0·25 % BEE (0·034 % anthocyanin,w/w) and 1·25 % BEE
(0·17 % anthocyanin, w/w) diets corresponded to estimated
anthocyanin doses of 20–40 mg and 100–200 mg per kg ofbody weight,
respectively. After 16 weeks, both BEE groups had significantly
lower liver weights, serum TAG, homoeostasis model assessmentand
serum monocyte chemoattractant protein-1 compared with HFD. The
0·25 %-BEE also had lower serum insulin and TNFα compared withHFD.
Hepatic fatty acid synthase mRNA was lower in both BEE groups,
whereas PPARγ2 mRNA and liver cholesterol were lower in 1·25 %-BEE,
suggesting decreased hepatic lipid synthesis. Higher adipose PPARγ
mRNA, transforming growth factor β mRNA and adipose tissuehistology
suggested a pro-fibrogenic phenotype that was less inflammatory in
1·25 %-BEE. Skeletal muscle mRNA expression of the myokineIL-6 was
higher in 0·25 %-BEE relative to HFD. These results suggest that
BEE may have improved some metabolic disturbances present in
thismouse model of obesity by lowering serum TAG, inflammatory
markers and insulin resistance.
Key words: Black elderberry extract: Obesity: Insulin
resistance: Diet-induced obese mice: Inflammation: Molecular
nutrition
According to the WHO, estimated worldwide rates ofoverweight and
obesity are 39 and 13 %, respectively(1). Obeseindividuals have
shortened life expectancies(2); however, theydo not typically die
of obesity itself but rather obesity-relatedcomorbidities, such as
CVD, diabetes and certain typesof cancers(3,4). The adipose
dysfunction and excessive ectopiclipid accumulation in tissues in
obesity promotes aninflammatory state that is thought to be an
underlying cause ofthese obesity-related comorbidities(5,6).
Therefore, methods thattarget and lower inflammation may be
effective at preventingobesity-related comorbidities. Anthocyanins,
a class ofpolyphenol belonging to the flavonoid family, are
dietarybioactives whose intake has been shown to be
inverselyassociated with inflammation and insulin resistance
inhumans(7). Cyanidin 3-glucoside (C3G), a major anthocyanin
innature, has been shown to enhance adipocyte function andprotect
adipocytes from metabolic stress in vitro, by enhancingPPARγ
activity and inhibition of forkhead box O1(8–10). Inanimal models,
dietary C3G fed at 0·2 % of the diet (w/w) for5 weeks has been
shown to improve insulin sensitivity and
adipose tissue inflammation in diet-induced obese
C57BL/6mice(11) and genetically diabetic mice(11,12). Purified
anthocyaninshave also displayed protective effects against hepatic
steatosis(11)
and non-alcoholic steatohepatitis(13) in mouse models. Inhuman
clinical studies, purified anthocyanin supplementation(300–320mg/d)
has been shown to improve systemic markersof inflammation in
healthy(14) and hypercholesterolaemic adults(15)
compared with placebo. However, humans do not consumepurified
anthocyanins in isolation, and there is evidence ofsynergistic
effects of different anthocyanins when used incombination in
vitro(15). Therefore, it is crucial to
investigateobesity-protective effects of anthocyanins from whole
foods,extracts and as isolated compounds.
Black elderberry (Sambucus nigra) contains one of the
highestanthocyanin contents reported in foods (1316mg/100 g
freshweight)(16). The major anthocyanins present in black
elderberryinclude C3G and cyanidin 3-sambubioside(17,18). Black
elderberryis commonly consumed in European cultures in wines and
otherprocessed beverages(19), with similar products available in
theUSA. The berry has been used for centuries in traditional
* Corresponding author: C. N. Blesso, fax +1 860 486 3674, email
[email protected]
Abbreviations: BEE, black elderberry extract; C3G, cyanidin
3-glucoside; CLS, crown-like structures; HFD, high-fat diet;
HOMA-IR, homoeostasis modelassessment of insulin resistance; LFD,
low-fat diet; LPL, lipoprotein lipase; MCP-1, monocyte
chemoattractant protein-1; TGFβ, transforming growth factor β.
British Journal of Nutrition (2015), 114, 1123–1131
doi:10.1017/S0007114515002962© The Authors 2015
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medicine throughout European and native American
cultures(19);however, research on its therapeutic potential is
limited.Therefore, we investigated the effects of anthocyanin-rich
blackelderberry extract (BEE) on the metabolic disturbances
asso-ciated with obesity using the high-fat diet (HFD)-induced
obeseC57BL/6J mouse model. We hypothesised that BEE feedingwould
attenuate the low-grade inflammation and insulin resistancein this
mouse model of obesity.
Methods
Animals and diets
HFD-fed C57BL/6 mice were used as a diet-induced model
ofobesity. Male C57BL/6J mice (8 weeks of age, n 56) were
obtainedfrom The Jackson Laboratory and allowed to acclimate tothe
animal facility for 2 weeks before being fed one of
fourexperimental diets for 16 weeks: low-fat diet control group
(LFD;10% energy as fat; n 8) (Harlan Teklad; TD.08806); HFD
controlgroup (60% energy as fat; n 16) (Harlan Teklad; TD.06414);
HFDwith 0·25% of BEE added by weight (0·25%-BEE; 60% energyas fat;
n 16); and HFD with 1·25% of BEE added by weight(1·25%-BEE; 60%
energy as fat; n 16). Spray-dried BEE (S. nigra)(standardised to
13% anthocyanins) on maltodextrin as anexcipient was kindly
provided by Artemis International. A singlebatch of BEE was
obtained from the supplier. BEE anthocyaninswere previously
characterised by HPLC and shown to be primarilyin the form of
cyanidin 3-sambubioside and C3G(18). The0·25%-BEE and 1·25%-BEE
diets corresponded to 0·034% antho-cyanin (w/w) and 0·17%
anthocyanin (w/w) in diets, respectively.The estimated anthocyanin
doses were 20–40mg (0·25%-BEE) and100–200mg (1·25%-BEE) per kg of
body weight after accountingfor weight gain throughout the study.
Carbohydrate content of dietscontaining BEE was adjusted to match
control HFD composition byreplacing maltodextrin with BEE. The
0·25%-BEE diet correspondsto a human dose of approximately 150mg of
anthocyanin/d,which can easily be achieved with regular consumption
of fruitssuch as berries(20). The 1·25%-BEE diet corresponds to
about1/2 serving (60 g) of black elderberry fruit(16)
(approximately740mg of anthocyanin/d) for a 70 kg person. Food
intake andbody weight were assessed weekly. Fresh diet was provided
tomice twice per week. After 16 weeks on experimental diets,
micewere fasted for 6–8 h before blood collection by cardiac
punctureand euthanasia. Blood was allowed to clot at room
temperature for30min before serum was isolated by centrifugation
(10 000 g for10min at 4°C) and then stored at −80°C. Tissues were
perfusedwith saline before being harvested, snap-frozen in liquid
N2 andstored at −80°C. Liver and adipose tissues were fixed in
10%neutral-buffered formalin for at least 48 h before
histologicalanalysis. All mice were housed in a
temperature-controlled roomand maintained in a 12 h light–12 h dark
cycle at the University ofConnecticut-Storrs vivarium. The Animal
Care and Use Committeeof the University of Connecticut-Storrs
approved all proceduresused in the current study.
Serum biochemical analysis
Total cholesterol, NEFA, TAG, glucose and alanine
amino-transferase (ALT) were measured using enzymatic assays,
as
described(18). Fasting insulin, monocyte
chemoattractantprotein-1 (MCP-1), IL-6, TNFα, adiponectin, resistin
and plas-minogen activator inhibitor (PAI) were measured by
Luminex/xMAP magnetic bead-based multiplexing assays using
MAGPIXinstrumentation (EMD Millipore). The homoeostasis
modelassessment of insulin resistance (HOMA-IR) equation was usedto
estimate insulin resistance based on fasting serum insulin
andglucose measurements(21).
Tissue lipid extraction and analysis
Hepatic lipids were extracted using methods
previouslyreported(18). Briefly, the lipids were extracted with
chloroform:methanol (2:1), dried under N2 at 60°C and solubilised
in TritonX-100 before being analysed for cholesterol and TAG
byenzymatic methods.
RNA isolation, cDNA synthesis and real-time
quantitativeRT-PCR
Total RNA was isolated from liver, skeletal muscle and
adiposetissues using TRIzol reagent (Life Technologies). RNA
wasthen DNase I-treated and reverse transcribed using the
iScriptcDNA synthesis kit (Bio-Rad). Gene expression was measured
byreal-time quantitative RT-PCR using SYBR Green (Bio-Rad) and
aCFX96 real-time PCR detection system (Bio-Rad). A detailedlist of
all primer sequences used in quantitative RT-PCR analysisis
provided in the online Supplementary material (onlineSupplementary
Table S1). Liver mRNA expression data werenormalised to the
geometric mean of the internal controlsglyceraldehyde 3-phosphate
dehydrogenase and ribosomalprotein large P0. The geometric mean of
glyceraldehyde3-phosphate dehydrogenase ribosomal protein large P0
andβ-actin was used as an internal control for adipose and
skeletalmuscle mRNA analysis. Expression relative to the
internalcontrols was determined using the 2�ΔΔCtmethod.
Histological analysis of tissues
Formalin-fixed liver and epididymal adipose tissue wereembedded
in paraffin and cut into 5 μm sections beforestaining. Liver
sections were stained with haematoxylin–eosin(H&E), whereas
adipose sections were H&E-stained orsubjected to Masson’s
trichrome staining to visualise connectivetissue. All histological
procedures were conducted at theConnecticut Veterinary Medical
Diagnostic Laboratory.The stained tissue sections were viewed under
bright-fieldmicroscopy at 200× magnification, and images were taken
withAxioCam ICc3 (Zeiss). The extent of macrophage infiltrationinto
adipose tissue was assessed by the manual counting ofcrown-like
structures (CLS) (three slides per animal) performedby a technician
blinded to group assignment.
Statistical analysis
One-way ANOVA was used to detect differences betweengroups with
post hoc multiple comparisons (Tukey’s test) whenappropriate (P<
0·05 deemed significant). GraphPad Prism
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version 6 software was used to conduct all statistical
analysis.Data are reported as mean values and standard error of
means.
Results
Effects of black elderberry extract on food intake andbody
weight
Although there were no differences in food intake (Fig.
1(A))between any of the four groups, the HFD groups had greaterbody
weight and weight change after 16 weeks than the LFDgroup (Fig.
1(B) and (C)). There were no differences in bodyweight or weight
change among the HFD groups. Liver weightswere approximately 13%
lower in both the 0·25%- and 1·25%-BEEgroups relative to the HFD
control group (Fig. 1(D)).
Black elderberry extract lowers serum TAG, inflammatorymarkers
and insulin resistance
Serum markers after 16 weeks are presented in Table 1. SerumTAG
was significantly reduced compared with HFD control inthe 0·25
%-BEE and 1·25 %-BEE groups by 25 and 30 %,respectively. There was
a significant increase in total serumcholesterol and ALT in the HFD
groups relative to LFD control;however, there were no differences
within the HFD groups. Nodifferences in serum NEFA were observed
between the groups.The HFD control group displayed significant
elevations inMCP-1, IL-6, TNFα, resistin and PAI-1, as well as a
reduction inadiponectin compared with LFD control (Fig. 2(A) and
(B)).BEE feeding attenuated the HFD-dependent increase inseveral
serum inflammatory cytokines/chemokines. There were
significant reductions in MCP-1 (−37 %) and TNFα (−47 %) withthe
0·25 %-BEE dose and a significant reduction in MCP-1(−30 %) with
the 1·25 %-BEE dose (Fig. 2(A)). However, BEEfeeding did not
attenuate the effects of HFD on serum IL-6,resistin, PAI-1 and
adiponectin (Fig. 2(A) and (B)). Fastingserum insulin was 32 %
lower in the 0·25 %-BEE group com-pared with HFD control (Fig.
2(C)), and HOMA-IR, a metric ofinsulin resistance, was
significantly lower in both BEE groupscompared with HFD control
(Fig. 2(D)).
Black elderberry extract reduces hepatic cholesterol
andlipogenic gene expression
HFD feeding significantly increased hepatic lipids comparedwith
LFD control, suggesting the development of hepaticsteatosis (Fig.
3(B) and (C)). This observation was confirmed byexamination of
H&E-stained livers, in which extensive lipiddroplet
accumulation was seen in the HFD groups (Fig. 3(A)).Hepatic lipid
accumulation with HFD appeared to beattenuated by BEE feeding (Fig.
3(C)). Hepatic cholesterol inthe 1·25 %-BEE group was significantly
lower (−32 %) than HFDcontrol (Fig. 3(B)). Hepatic TAG did not
differ significantlybetween HFD groups, although BEE groups tended
to belower (Fig. 3(C)). After hepatic lipid analysis, we
performedreal-time quantitative RT-PCR analysis to assess hepatic
mRNAexpression of lipid metabolism-related genes. Expression of
thelipogenic gene fatty acid synthase (Fas) was
significantlyreduced in both BEE-fed groups, whereas PPARγ2
wassignificantly reduced only in the 1·25 %-BEE group relative
tothe HFD control (Fig. 4), corresponding with histological
andbiochemical analysis.
4
3
2
1
0
4
3
2
1
0
LFD HFD 0.25 % 1.25 %
LFD HFD 0.25 % 1.25 % LFD HFD 0.25 % 1.25 %
1612840
60
50
40
30
20
10
0
30
20
10
0
Week
Weekly body weight
Weight change Liver weight
Live
r w
et w
eigh
t (g)
Mean daily food intake
Foo
d in
take
(g/
d)
Bod
y w
eigh
t (g)
Δ B
ody
wei
ght (
g)
a aaa
aa
b b b bc c
(A) (B)
(C) (D)
Fig. 1. Black elderberry extract (BEE) reduces liver weight with
no change in food intake or weight gain. Food intake (A) and body
weight of animals (B) weremeasured weekly. Mean weight change was
calculated after 16 weeks (C), and liver weight was measured at the
time of killing (D) (n 8–16/group). Values are meanswith their
standard errors represented by vertical bars. a,b,c Mean values
with unlike letters were significantly different using post hoc
comparisons (P
-
Black elderberry extract does not attenuate adipose
tissuemacrophage infiltration and fibrosis
Compared with the LFD group, all HFD groups had a
noticeableincrease in macrophage infiltration indicated by H&E
staining, withan increased number of CLS (Fig. 5(A) and (B)). There
were nodifferences in CLS among the HFD groups, as all groups
appearedto have extensive immune cell infiltration and large
pocketswithout adipocytes, suggesting fibrosis. The adipose
tissuebecame markedly fibrotic in the HFD groups, as shown
byMasson’s trichrome blue staining of connective tissue (Fig.
5(C)).Indeed, the large pockets that lacked adipocytes stained
stronglyfor connective tissue, and this staining appeared to be
greatest inthe 1·25%-BEE group relative to the other HFD groups. To
furtherexamine adipose tissue, real-time quantitative RT-PCR
wasperformed to examine changes in mRNA expression between
HFDgroups (Fig. 5(D)). In the 1·25%-BEE group, there were
significantincreases in expression of PPARγ and a PPARγ target
gene,adipocyte protein 2, compared with the HFD control group.
Another PPARγ target gene, lipoprotein lipase, was
significantlyhigher in the 1·25%-BEE group relative to the
0·25%-BEEgroup. F4/80, a macrophage marker, was also more
highlyexpressed in the 1·25%-BEE group compared with the HFDcontrol
and 0·25%-BEE groups. Interestingly, although the 1·25%-BEE group
had greater F4/80 mRNA expression, TNFα expressionwas significantly
reduced compared with the 0·25%-BEE group,suggesting that the
macrophages were less inflammatory. Theexpression of CD11c, a
phenotypic marker of ‘classically activated’M1-like inflammatory
macrophages, was not different between anyof the groups, suggesting
that 1·25%-BEE macrophages are ofthe M2-like ‘alternatively
activated’ phenotype that remodeladipose tissue and are not
inflammatory. Supporting this notion,mRNA expression of
transforming growth factor β (TGFβ), apro-fibrogenic cytokine, was
increased in the 1·25%-BEE grouprelative to the other HFD groups.
In addition, collagen VI α3(Col6a3), a downstream target of TGFβ
that is highly enriched inadipose tissue, was significantly
increased in the 1·25%-BEE grouprelative to the 0·25%-BEE
group.
Table 1. Serum markers of C57BL/6J mice after 16 weeks(Mean
values with their standard errors)
Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) TAG (mmol/l)
NEFA (mmol/l) ALT (U/l)
n Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM
LFD 8 3·43a 0·11 2·26a 0·13 0·37a 0·06 0·62 0·05 15·93a 2·71HFD
16 4·88b 0·26 3·42b 0·20 0·55b 0·03 0·66 0·03 43·21b 3·130·25%-BEE
16 4·65b 0·24 3·11b 0·18 0·41a 0·03 0·66 0·03 39·36b 3·161·25%-BEE
16 4·69b 0·18 3·34b 0·20 0·38a 0·05 0·60 0·04 41·26b 4·08
ALT, alanine aminotransferase; BEE, black elderberry extract;
HFD, high-fat diet; LFD, low-fat diet.a,b Mean values within a
column with unlike superscript letters were significantly different
using post hoc comparisons (P
-
Black elderberry extract alters lipid metabolism-relatedgene
expression but does not attenuate skeletal muscleinflammation
To examine skeletal muscle inflammation and metabolicfunction,
mRNA expression in quadriceps was measured(Fig. 6). Compared with
LFD control, HFD feeding resulted inhigher mRNA expression of
skeletal muscle MCP-1 and themacrophage marker, CD68, suggesting
that the obese statecaused inflammation in this tissue. BEE feeding
was unable toalter MCP-1 or CD68 expression compared with HFD
control.There was also a marked increase in the expression of
lipid
metabolism-related genes acyl-CoA dehydrogenase, Lpl andacyl-CoA
oxidase (Acox) with high-fat feeding compared withLFD. Lpl and Acox
expression were decreased significantly inthe 1·25 %-BEE group
relative to the HFD control. IL-6, amyokine and regulator of
substrate utilisation, was increased by2·7-fold in the 0·25 %-BEE
group compared with the HFDcontrol group.
Discussion
Targeting the excessive lipid accumulation and inflammationin
obesity may lead to successful therapies that reduce theprevalence
of obesity-related comorbidities(5). Anthocyaninsare dietary
bioactives that have been shown to reduceinflammation and insulin
resistance in obese animals(11,12).Black elderberry is a berry rich
in anthocyanins, but there islimited research examining its effects
on inflammation inchronic disease models. In this study, C57BL/6J
mice fed BEEwere shown to have an attenuation of insulin resistance
andsystemic inflammation compared with HFD controls. BEE-fedmice
were also shown to have lower serum TAG and modestreductions in
hepatic lipids compared with HFD controls. Thesechanges suggest
that BEE may have potential in amelioratingthe lipotoxicity and
inflammation present in obesity.
Anthocyanins have been shown to have limited bioavail-ability
and are not found in the serum in significant quantities(
-
the form of metabolites. C3G is degraded to protocatechuicacid
(PCA), either spontaneously or after catabolism by thegut
microbiota, which can then be absorbed and enter
thebloodstream(25). Compared with intact C3G, much higher
con-centrations of these phenolic degradation products and
theirphase II conjugates were found in serum, urine and faeces
inhuman subjects after 13C-labelled C3G ingestion(24). In this
case,it is likely that the metabolites of anthocyanins are
primarilyresponsible for their physiological effects.In the current
study, both groups of BEE-fed mice were found
to have improvements in many serum markers of obesity-related
metabolic complications. BEE-fed mice had 25–30 %reductions in
fasting serum TAG compared with HFD control,although no other
significant differences were observed inserum lipids. BEE appeared
to attenuate systemic inflammation,
as mice fed the lower dosage of BEE had >30 % lower
serumMCP-1 and TNFα, whereas only MCP-1 reached significance inthe
higher dosage group. Both BEE-fed groups displayed 20and 40 %
reductions in fasting insulin and HOMA-IR, respec-tively, which
suggests that BEE reduced insulin resistance inthese obese mice.
However, future studies should examineinsulin resistance using
glucose tolerance tests or clamps, asthese would provide greater
insight into the metabolic effects ofBEE in obese conditions.
BEE-fed mice also displayed modest changes in markers ofhepatic
steatosis that increase with diet-induced obesity. BEE-fedmice were
found to have 13% lower liver weights compared withHFD control. We
observed dose-related effects of BEE on hepaticlipids and lipogenic
gene expression. There appeared to be amodest attenuation of
hepatic steatosis in the 1·25%-BEE group
Skeletal muscle mRNA
GLUT4 ACOX ACAD CD68 LPL IL-6 MCP-1
50
40
30
20
10
0
Rel
ativ
e ex
pres
sion
a a a aa
aaab
b b
b
b
b
b b b
b
bb ba,ba,b
a,b
a,b
b,c
c
c
Fig. 6. Effect of black elderberry extract (BEE) on skeletal
muscle gene expression. Skeletal muscle mRNA was measured by
real-time quantitative RT-PCR. Datawere normalised to endogenous
reference gene expression (n 8–16/group). ACAD, acyl-CoA
dehydrogenase; ACOX, acyl-CoA oxidase 1; CD68, cluster
ofdifferentiation 68; HFD, high-fat diet; LFD, low-fat diet; LPL,
lipoprotein lipase; MCP-1, monocyte chemoattractant-1. Values are
means with their standard errorsrepresented by vertical bars. a,b,c
Mean values with unlike letters were significantly different using
post hoc comparisons (P< 0·05). , LFD; , HFD; , 0·25% BEE;,
1·25% BEE.
Adipose crown-like structures
Adipose mRNA
a
bb
b
LFD
LFD
HFD
HFD
0.25 %
0.25 %
1.25 %
1.25 %
LFD HFD
0.25 % 1.25 %
15
10
5
5
4
3
2
1
0
0
Rel
ativ
e ex
pres
sion
Num
ber
of C
LS (
/200
x H
PF
)
GLUT
4
Adipo
Q
PPAR
�aP
2LP
LF4
80TN
F�
MCP
-1
CD11
c
TGF�
Col6a
3
a aa a a
a
ba
a,ba
a
a
b
a
aa
a a
a
aa
b
a
b
b
b
a,b
b
a,ba,b a a,b
a,b
(A) (B)
(C) (D)
Fig. 5. Effect of black elderberry extract (BEE) on adipose
tissue inflammation and fibrosis. Epididymal adipose
haematoxylin–eosin (H&E) (A) and Masson’strichrome (C) staining
was performed as described in the Methods section. Crown-like
structures (CLS) were manually counted from adipose H&E stains
and averagedacross three random 200× high-powered fields (HPF) (B)
(n 8/group). Adipose mRNA expression was measured by real-time
quantitative RT-PCR. Data werenormalised to endogenous reference
gene expression (D) (n 16/group). AdipoQ, adiponectin; aP2,
adipocyte protein 2; CD11c, integrin, αX (complement component3
receptor 4 subunit); Col6a3, collagen, type VI, α3; F480, EGF-like
module-containing mucin-like hormone receptor-like 1; HFD, high-fat
diet; LFD, low-fat diet;LPL, lipoprotein lipase; MCP-1, monocyte
chemoattractant-1; TGFβ, transforming growth factor-β. Values are
means with their standard errors represented by verticalbars. a,b
Mean values with unlike letters were significantly different using
post hoc comparisons (P< 0·05). , HFD; , 0·25% BEE; , 1·25%
BEE.
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compared with the HFD control, with noticeably smaller
lipiddroplets, suggesting less macrosteatosis. A significant
reduction ofhepatic cholesterol by 32% and a non-significant 16%
attenuationin hepatic TAG in the 1·25%-BEE group confirm this
observation.These changes may be explained in part by reductions in
hepaticFAS and PPARγ2 mRNA in the 1·25%-BEE group. No changes
inβ-oxidation-related gene expression were observed among theHFD
groups, suggesting that BEE influenced TAG synthesis ratherthan
oxidation.The effects of BEE feeding on adipose tissue did not
appear to
explain the differences in serum markers between HFD
groups.Despite no changes in the epididymal adipose CLS count
amongthe HFD groups, the 1·25%-BEE displayed a 211% increase in
themRNA expression of the pan-macrophage marker F4/80.Although
F4/80 expression was increased, the 1·25%-BEE groupadipose tissue
did not appear inflamed, having significantly lowerTNFα expression
than the 0·25%-BEE group, and this approachedsignificance compared
with HFD control. Furthermore, CD11c, amarker of pro-inflammatory
‘classically activated’ M1-likemacrophages, was not increased in
the 1·25%-BEE group.Adipose mRNA expression of PPARγ and TGFβ,
which areinduced in M2-like ‘alternatively activated’
macrophages(26,27),was significantly increased in the 1·25%-BEE
groups comparedwith HFD control. Potentially, the macrophages in
the 1·25%-BEEadipose tissue were more of an M2-like phenotype,
where theywould be pro-resolving and anti-inflammatory. Expression
levelsof PPARγ target genes (aP2, LPL) were increased in
1·25%-BEEadipose relative to other HFD groups, suggesting that
PPARγactivity was increased. PPARγ activation in macrophages has
beenshown to induce an anti-inflammatory M2-like phenotype
thatimproves metabolic function in obese mice(26,28).
Alternativelyactivated M2-like macrophages have also been shown
tostimulate fibrogenesis in vitro via production of
TGFβ(29).Connective tissue staining of sectioned adipose tissue
appearedto be somewhat greater in the 1·25%-BEE group, which
alsodisplayed a higher expression of Col6a3, a fibrogenic gene
thatwas shown to be induced downstream of TGFβ signallingin
vitro(30). In human obese subjects, adipose tissue macrophagesthat
were present in fibrotic areas and not in CLS were shown tobe
primarily M2-like macrophages, which produce TGFβ andincrease
adipocyte collagen VI(27). Thus, despite the stronginduction of
F4/80 mRNA in the 1·25%-BEE adipose, theinfiltrated macrophages do
not appear to be inflammatory andmay be of the M2-like
anti-inflammatory and pro-fibrogenicphenotype.In skeletal muscle,
we observed dose-related effects of
BEE on the expression of several fatty acid metabolism
genes.Skeletal muscle mRNA expression levels of LPL and ACOX
werereduced by 50 and 46 % in the 1·25 %-BEE group comparedwith HFD
control, which indicates major changes in fatty acidmetabolism.
However, these genes were highly induced in allHFD groups compared
with LFD, suggesting that thesedecreases with the 1·25 %-BEE dose
were indicative of anattenuation of the HFD effect. This may
possibly be explainedby reduced fatty acid availability to the
skeletal muscle as aconsequence of greater adipose PPARγ activation
and adiposefatty acid buffering capacity. With long-term HFD
feedingin mice (>12 weeks), skeletal muscle can exhibit
extensive
macrophage infiltration and inflammation similar to
adiposetissue(31). BEE feeding was unable to attenuate the
apparentmacrophage infiltration (>30-fold increases in CD68
mRNAexpression) and inflammation with HFD feeding
(>five-foldincreases in MCP-1 mRNA expression). Of interest was
the2·7-fold increase in IL-6 mRNA expression in the 0·25 %-BEEgroup
compared with HFD control. IL-6 is a myokine andsomewhat
controversial in regard to its effects in skeletalmuscle, systemic
glucose tolerance, and insulin resistance(32,33).Although
traditionally viewed as an inflammatory cytokine thatincreases
hepatic insulin resistance, increasing evidence pointsto a
beneficial metabolic role in promoting glucose uptakeand fatty acid
oxidation in skeletal muscle(34,35). This may besignificant in
regulating systemic glucose tolerance, as theskeletal muscle is the
major organ involved in whole-bodyglucose disposal in
humans(36).
Our findings are consistent with other reports that havefed
anthocyanins and anthocyanin-rich foods to obese rodentmodels. Guo
et al.(11) observed significant reductions in plasmainsulin,
HOMA-IR, MCP-1 and TNFα, with no changes in IL-6 afterfeeding
diet-induced obese C57BL/6 mice with 0·2% (w/w)C3G for 5 weeks. We
reported reductions in fasting insulin andHOMA-IR by 20–30 and 40%,
respectively. DeFuria et al.(37) alsoobserved decreases in insulin
resistance in C57BL/6J mice fedblueberry powder in the diet at 4 %
by weight (about 0·12%anthocyanins) for 8 weeks. Similarly, Chuang
et al.(38) reportedan improvement in glucose tolerance in
diet-induced obeseC57BL/6 mice fed 3% (w/w) grape powder for 18
weeks. Thesignificant reductions in serum MCP-1 (37 and 30% in
0·25%-BEEand 1·25%-BEE groups, respectively, relative to HFD
control)and TNFα (47 % in the 0·25%-BEE group relative to HFD
control)are comparable to the reductions of 30–50% reported
byChuang et al.(38) in serum inflammatory markers. Therefore,
theresults that we report in this study are consistent with
previousfindings on the efficacy of anthocyanin-rich foods in
amelioratinginflammation and insulin resistance in obese rodent
models.
In conclusion, BEE-fed mice had reduced serum
inflammatorymarkers and insulin resistance, as measured by HOMA-IR.
BEE-fedmice had lower fasting TAG and modest reductions in
hepaticlipids, possibly explained by reductions in hepatic FASand
PPARγ2. Despite a lack of difference in CLS in the HFDgroups, the
1·25%-BEE-fed mice appeared to have moremacrophage infiltration in
the adipose tissue, as demonstratedby greater F4/80 mRNA
expression. These macrophages do notappear to be inflammatory,
however, and may be depositingmore collagen. Across the tissues,
changes in mRNA expressionin the BEE-fed groups suggest differences
in fatty acid metabolismincluding potentially decreased lipogenesis
in the liverand increased adipogenesis in the adipose tissue. Both
BEEdoses appear to attenuate some of the complications induced
byHFD feeding, although the 1·25% (w/w) dose does not appear
toimprove upon the serum changes observed with the 0·25% (w/w)dose
and may even cause complications in the adipose becauseof
fibrogenic effects. Overall, BEE appeared to attenuatesystemic
inflammation and insulin resistance that occurs withdiet-induced
obesity in this mouse model, but further research iswarranted on
black elderberry consumption and effects inhumans.
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Acknowledgements
This work was supported by USDA AFRI (CONS2014-06619)and USDA
Hatch (CONS00934) grants to C. N. B.N. F. conducted the research,
analysed the data and wrote the
paper; G. N. conducted the research and provided input for
thepaper; J. R., C. M. P. and C. J. conducted the research; C. N.
B.designed the research, analysed the data and had
primaryresponsibility for final content. All authors read and
approvedthe final manuscript.There are no conflicts of
interest.
Supplementary material
For supplementary material/s referred to in this article,
pleasevisit http://dx.doi.org/doi:10.1017/S0007114515002962
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Black elderberry extract attenuates inflammation and metabolic
dysfunction in diet-induced obesemiceMethodsAnimals and dietsSerum
biochemical analysisTissue lipid extraction and analysisRNA
isolation, cDNA synthesis and real-time quantitative
RT-PCRHistological analysis of tissuesStatistical analysis
ResultsEffects of black elderberry extract on food intake and
body weightBlack elderberry extract lowers serum TAG, inflammatory
markers and insulin resistanceBlack elderberry extract reduces
hepatic cholesterol and lipogenic gene expression
Fig. 1Black elderberry extract (BEE) reduces liver weight with
no change in food intake or weight gain. Food intake (A) and body
weight of animals (B) were measured weekly. Mean weight change was
calculated after 16weeks (C), and liver weight was measureBlack
elderberry extract does not attenuate adipose tissue macrophage
infiltration and fibrosis
Table 1Serum markers of C57BL/6J mice after 16weeks(Mean values
with their standard errors)Fig. 2Black elderberry extract (BEE)
reduces serum inflammation and insulin resistance. Serum
cytokines/chemokines, adipokines and insulin were examined by
multiplexing assays (A–C). homoeostasis model assessment of insulin
resistance (HOMA-Black elderberry extract alters lipid
metabolism-related gene expression but does not attenuate skeletal
muscle inflammation
DiscussionFig. 4Black elderberry extract (BEE) reduces lipogenic
mRNA expression in the liver. Hepatic mRNA expression was measured
by real-time quantitative RT-PCR. Data were normalised to
endogenous reference gene expression (n 8–16/group). ACAD, acFig.
3Effect of black elderberry extract (BEE) on hepatic lipids and
steatosis development. Liver haematoxylin–eosin (H&E) histology
was performed as described in the Methods section (A) (n 8/group).
Hepatic lipids were extracted withFig. 6Effect of black elderberry
extract (BEE) on skeletal muscle gene expression. Skeletal muscle
mRNA was measured by real-time quantitative RT-PCR. Data were
normalised to endogenous reference gene expression (n 8–16/group).
ACAD, acyl-CoFig. 5Effect of black elderberry extract (BEE) on
adipose tissue inflammation and fibrosis. Epididymal adipose
haematoxylin–eosin (H&E) (A) and Masson’s trichrome (C)
staining was performed as described in the Methods section.
Crown-lAcknowledgementsACKNOWLEDGEMENTSReferencesReferences